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*Correspondence: P. A. Marinho. Instituto de Criminalística de Minas Gerais. Rua Juiz de Fora, 400 - Bairro Barro Preto - 30180-060 - Belo Horizonte - MG, Brasil. E-mail: pabloalvesmarinho@yahoo.com.br Article Brazilian Journal of Pharmaceutical Sciences vol. 46, n. 4, out./dez., 2010
Quantification of LSD in illicit samples by high performance liquid
chromatography
Pablo Alves Marinho1,*, Edna Maria Alvarez Leite^2
(^1) Institute of Criminology of Minas Gerais, Belo Horizonte, 2 Toxicology Laboratory, Pharmacy College, Federal University of Minas Gerais In the present study, a method using high performance liquid chromatography to quantify LSD, in blotter papers seized in Minas Gerais, was optimized and validated. Linearity, precision, recovery, limits of detection and quantification, and selectivity were the parameters used to evaluate performance. The samples were extracted with methanol:water (1: 1) in an ultra-sound bath. The linearity between 0. and 20.00 μg/mL (0.5 and 200.0μg of LSD/blotter) was observed with satisfactory mean intra and inter assay precision (RSDr = 4.4% and RSDR = 6.4%, respectively) and with mean recoveries of 83.4% and 84.9% to the levels of 1.00 and 20.00 μg/mL (10 and 200μg LSD/blotter). The limits of detection and quantification were 0.01 and 0.05 μg/mL, respectively (0.1 and 0.5 μg of LSD/blotter). The samples of blotters (n =22) were analyzed and the mean value of 67.55 μg of LSD/blotter (RSD=27.5%) was found. Thus, the method used showed satisfactory analytical performance, and proved suitable as an analytical tool for LSD determination in illicit samples seized by police forces. Uniterms: LSD/detection. Forensic chemistry. Analytical toxicology. In-house validation. High Performance Liquid Chromatography/quantitative analysis. No presente trabalho, um método utilizando cromatografia líquida de alta eficiência foi otimizado e validado para quantificar o LSD em selos apreendidos em Minas Gerais. A linearidade, precisão, recuperação, limites de detecção e quantificação e seletividade foram os parâmetros de desempenho avaliados. As amostras foram extraídas com metanol: água (1:1) em banho de ultra-som. A linearidade entre 0,05 a 20,00 mg/mL (0,5 a 200 μg LSD/blotter) foi observada com precisão média, intra e inter ensaio, satisfatória (RSDr = 4,4% e RSDR = 6,4%, respectivamente) e com recuperações médias de 83,4% e 84,9% para os níveis de LSD de 1,00 e 20,00 mg/mL (10 e 200 μg LSD/selo). Os limites de detecção e quantificação encontrados foram de 0,01 e 0,05 mg/mL, respectivamente (0,1 e 0,5 μg LSD/selo). As amostras de selos (n = 22) foram analisadas e o valor médio encontrado foi de 67,55 μg de LSD/selo (RSD% = 27,5). Desta forma, o método analítico apresentou desempenho satisfatório, capaz de ser utilizado como instrumento de análise para a determinação do LSD em amostras ilícitas apreendidas pelas forças policiais. Unitermos : LSD/detecção. Química Forense. Toxicologia analítica. Validação intralaboratorial. Cromatografia líquida de alta efeciência/análise quantitativa.
INTRODUCTION
The Swiss chemist Albert Hofmann first synthesized
LSD (lysergic acid diethylamide) in 1938. The short form
LSD comes from its early code name LSD-25 , which is an
abbreviation for the German “Lysergsäure-diethylamid”
followed by a sequential number (NIDA, 2001; Cashman,
LSD chemical structure is shown in Figure 1.
Lysergic acid diethylamide commonly known as
acid, lysergide or sweet is a semisynthetic drug made from
lysergic acid, an alkaloid produced by fungus Claviceps
purpurea (Oga, 2003; Schiff, 2006).
Different forms of LSD are sold on the illicit market
696 P. A. Marinho, E. M. A. Leite
such as sugar cubes, paper dosage units (blotter papers),
small tablets (microdots), and gelatin matrix containing
LSD that is solidified and cut into square pieces called
windowpanes (United Nations, 2003, 1989).
Research carried out in Brazil in 2005, by the
Centro Brasileiro de Informações sobre Drogas Psico-
trópicas- CEBRID (Brazilian Center for Information on
Psychotropic Drugs) and Secretaria Nacional Antidrogas
- SENAD (National Anti-drugs Secretariat) showed that
1.1% of Brazilians have used hallucinogens at least once
in their lifetime. These rates are lower than those found in
the survey carried out by the Substance Abuse and Mental
Health Services Administration SAMHSA (14.3%) (Car-
lini et al ., 2005).
Brazilian law prohibits the use of LSD and its
isomers in the country (Resolution 344/1998 of Agência
Nacional de Vigilância Sanitária-ANVISA) but statistics
compiled by the Observatório Brasileiro de Informações
sobre Drogas-OBID showed an increase (539 %) in LSD
seizure by National Police from 2004 to 2006. Thus, it is
very important to identify the drugs seized.
Ultraviolet (UV) irradiation, color tests using
Ehrlich’s reagent, reagent of Mandelin or Marquis and
thin layer chromatography (TLC) with fluorescent addi-
tive in the stationary phase for instance, can all be used to
screen for LSD in illicit samples (United Nations, 1989;
Moffat, 2004).
Usual techniques for quantification of LSD are high
performance liquid chromatography with fluorescence,
ultraviolet or mass spectrometry detection, as well as gas
chromatography and capillary electrophoresis with mass
spectrometry detection. (Chung et al. , 2009; Shutter et al. ,
2009; Favretto et al. , 2007).
Unfortunately, few laboratories of toxicology in
Brazil and other developing countries are equipped to
follow international protocols. The lack of equipment for
mass spectrometry in these laboratories can be explained
by the high cost of acquisition and maintenance of such
equipment (GGLAS , 2008). Moreover, there are no studies
on the quantitative profile of LSD in illicit samples seized
in Brazil and this gap precludes intra- and inter-regional
comparative studies on these samples.
Thus, the aim of the present study was to optimize
and validate an analytical method for LSD determination
in samples of blotter papers traded illicitly, using high
performance liquid chromatography with an ultraviolet
detector (HPLC-UV) and determination of the quantitative
profile of the analyzed samples.
MATERIAL AND METHODS
Instrument
Chromatographic analysis was performed by a
Hewlett Packard®^ HPLC, model HP 1200 Series, equipped
with isocratic pump, ultraviolet detector and ChemStation
Rev.B.02.01 software (Agilent Technologies 2001-2006).
A Zorbax Eclipse XDB - C8, 5 μm (150 mm x 4.6 mm)
column (Agilent Technologies®) was used.
Standards and reagents
Lysergic acid diethylamide (LSD) – 1.0 mg/mL;
Lysergic acid methylpropylamide (LAMPA) – 1.0 mg/
mL; (-) D^9 -Tetrahydrocannabinol (THC) – 1.0 mg/mL,
were used, all obtained from Cerilliant Corporation®
(Texas, U.S.A.). Trade standards of caffeine (Synth®),
benzocaine (Sigma-Aldrich®), lidocaine (Sigma- Aldri-
ch®) and cocaine extract were purified by our laboratory.
Acetonitrile and methanol grade HPLC (J.T. Baker®);
acid acetic (Vetec®) and ammonium carbonate (Reagen®)
grade PA was used.
Samples
Samples of blotters came (n=22) from seizures car-
ried out by the police of Minas Gerais State between 2006
and 2009 and were sent to the Institute of Criminology
of Minas Gerais in order to be examined by chemical-
toxicological assay. The blotters had an average mass of
17 μg and dimensions of 7 x 7 mm, as shown in Figure 2.
METHODS
Sample preparation
The samples were extracted with 2.0 mL of me-
thanol: water (1:1) for twenty minutes in an ultrasonic
bath, changing the solvent extractor every 5 minutes, in
order to extract the maximum LSD impregnated in the
blotters.
FIGURE 1 - LSD chemical structure (P.M.: 323.432; CAS: 50-37-3).
698 P. A. Marinho, E. M. A. Leite
Methanol:water (1:1) was selected as the LSD sol-
vent extractor according to the study by Veress (Veress,
1993). Blotters are usually impregnated with LSD in
salt form (for example, LSD tartrate) so a high polarity
solvent is more efficient to extract the drug. The results
of the present study were consistent with those found by
Veress 1993, as LSD levels from blotters extracted with
methanol were significantly lower than those obtained
using methanol:water (1:1) extraction.
Before examining the assumptions related to the li-
near regression, the presence of dispersed values (outliers)
was analyzed by the Jacknife standardized residuals test.
Only one outlier value was detected. Figure 4 shows a
graph of residuals without the outlier value, which was
removed earlier.
The presence of heteroscedasticity or the lack of
adjustment to the linear model was not evident visually.
Ryan-Joiner, 1976, Brown- Forsythe, 1974 and
Durbin-Watson 1951 tests were applied in order to verify
the normality, homoscedasticity and independence of the
regression residues, respectively (Table I.)
The linear range obtained in this study after the
exclusion of outlier and linear regression assumptions
evaluation was from 0.05 to 20.0 μg/mL (0.5 to 200.0 μg
of LSD/blotter). Figure shows the LSD linearity curve.
LD and LQ values were 0.01 and 0.05 μg/mL, respec-
tively (equivalent to 0.1 and 0.5 μg of LSD/blotter). Both
of these presented adequate S/N ratio and LQ also showed
satisfactory precision (8%). According to Thompson, 2004,
the RSD under reproducibility conditions changes with the
compound concentration in the sample, whereby lower
concentrations show higher acceptable RSDs.
The precision under repeatability and day to day,
attained in the present study are shown in Table II, and
FIGURE 3 - Amount of LSD extracted from five LSD blotter papers in each cycle. FIGURE 4 - Residual plots for outlier treatment by Jacknife standardized residuals test, demonstrating maximum and minimum allowed intervals. The horizontal lines correspond to ± (t (^) 0.975; n-2) sres. FIGURE 5 - Linear range of HPLC-UV method for LSD determination, demonstrating curve equation and determination coefficient. TABLE I - Statistical parameters for linear regression evaluation Statistic Value N 14 Normality R 0. P p>0. Homoscedasticity tL 0. P 0. Independence D 1. P p>0. n: data number; R: Ryan-Joiner correlation coefficient; tL: Levene statistic t; p:significance; D: Durbin-Watson statistic
Quantification of LSD in illicit samples by high performance liquid chromatography (^699) TABLE II - Mean and intra and inter assay relative standard deviation precision at different levels of LSD Concentration (μg/mL) Amount/blotter (μg) Precision intra assay (n=5) RSD (%) Precision inter assay (n=5) RSD (%) 0.05 0.5 4.75 8. 10.0 100 4.51 6. 20.0 200 3.84 4. TABLE III - Mean recovery at two different levels of concentration and respective relative standard deviation Concentration (μg/mL) Amount/blotter (μg) Mean Recovery RSD (%) 1.0 10 83.84 2. 20.0 200 84.85 7.
the results are in agreement with Pocklington, 1990 (11%
and 23%).
Percentage recovery found for the two different
concentrations studied can be seen in Table III. These data
are in accordance with those reported by the European
Commission, 2002, that cites acceptable recovery values
as lying in the range between 80 to 110% for substances at
concentrations higher than 0.01 ppm in the sample.
No substance used to verify the method selectivity
showed a retention time able to interfere with the LSD
chromatographic peak (Figures 6, 7 and 8).
Twenty-two (22) blotter samples were analyzed and
quantified by the method validated. For LSD quantifica-
tion, a calibration curve was prepared with three levels of
FIGURE 6 - Chromatogram obtained after injection of standards of caffeine (tr = 1.92 min), benzocaine (tr = 4.33 min), LSD (tr = 7.64 min), cocaine (tr = 12.77 min) and lidocaine (tr = 23.51 min).
concentration, in triplicate, using “blank blotters” as ex-
plained in the recovery study described above. The results
are shown in Table IV. Figure 9 shows a chromatogram of
an analyzed LSD blotter sample.
Other “blank blotter” papers were previously im-
pregnated on their external surface with the same LSD
quantity and then dried and submitted to the extraction
method for recovery calculation.
The amount of LSD determined in the analyzed
samples was consistent with the values found in literature,
lying within the range described by the literature which
reports a wide variation of LSD in blotters (30 to 500 μg)
analyzed by different authors (Veress, 1993; United Na-
tions, 1989; Clarkson et al. , 1998).
Quantification of LSD in illicit samples by high performance liquid chromatography (^701) TABLE IV - LSD in 22 blotters seized by Police of Minas Gerais state LSD (μg) Blotters 31.51 17 33.91 19 51.02 18 53.36 2 53.72 4 54.43 1 55.08 3 55.34 21 57.58 20 58.72 5 62.79 15 65.07 22 76.25 16 78.11 8 81.69 12 81.98 6 85.10 11 86.82 10 87.63 7 88.58 9 92.88 13 94.60 14 Mean value: 67.55± 18.61 (CV= 27.5%) FIGURE 9 - Chromatogram after injection of blotter extract in methanol: water (1:1) tr LSD = 7.86 min.
LSD in illicit samples. Further similar studies carried out
in the future with samples seized in other states, will allow
a quantitative profile of the drug in these samples to be
defined at the national level.
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